Influenza is one of the oldest and most common diseases known to man causing between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world. Also swine are susceptible to human and avian influenza virus since they posses both receptors in their respiratory tract. Thus, swine get infection and pneumoni from human influenza strains and may serve as a dangerous mixing vessel for generation of new recombinant influenza strains with pandemic potential.
Influenza rapidly spreads in seasonal epidemics affecting 5-15% of the population and the burden on health care costs and lost productivity are extensive (WHO). Influenza like illness was first described by Hippocrates in the year 412 BC. Up to the 19th century influenza was thought to be a bacterial infection. Virus as the causative agent was first determined in 1931 by Richard Shope. The first known influenza A pandemic was in 1580 and since then there has been 31 pandemics of which three appeared in the 20th century namely the ‘Spanish flu’ in 1918, the ‘Asian flu’ in 1957 and the ‘Hong Kong flu’ in 1968, respectively. The pandemic of 1918 influenza A H1N1 was the worst pandemic in newer times causing 20 to 50 million deaths worldwide. The most common form of influenza is seasonal outbreaks and epidemics of variable severity.
Zoonosis of avian influenza virus (AIV) able to infect humans and swine and the spread in Asia, parts of Europe and the Middle East has recently evoked the concern about a pandemic occurring also in the 21st century. The causative strain of the pandemic will probably be unknown until the pandemic emerges and there will be an urgent need for a vaccine. Therefore fast diagnosis and characterisation of circulating strains as well as emerging strains, new alternative vaccines approaches and production ways will be required in order to minimise the severity of the pandemic. Since seasonal influenza A vaccines are also produced on eggs an epidemic of highly pathogenic AIV among poultry will also influence the production of seasonal vaccines. Moreover the traditional influenza protein vaccines only have a limited protective effect. Also seasonal vaccines has to be changed every season because of the genetic drift of influenza A virus and the narrow type specific antibody induction by traditional influenza A protein vaccines. Therefore there is a need for new alternative influenza A vaccines with different properties
The influenza virus belongs to the Orthomyxoviridae family. The family includes three genera; influenza A, B and C viruses, identified by antigenic differences in their nucleoprotein (NP) and matrix protein (M). The influenza A genus is further divided into subtype combinations based on the antigenic differences of the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA). The A strain have evolved to be able to infect several other mammalian species (e.g. horses and swine). Influenza A viruses of all recognised 16 HAs and 9 NAs antigenic subtypes have been recovered from aquatic birds but few infect other animal species indicating that aquatic birds are the natural reservoirs of influenza A.
The influenza A viruses have been the causative agents for the major pandemics and most of the annual outbreaks of epidemic influenza. This invention solely focuses on the influenza A genus. The current nomenclature system for human influenza viruses includes the geographical location of first isolation, strain number, and year of isolation. The antigenic description of HA and NA is given in brackets, e.g. A/Moscow/10/99(H3N2). Nonhuman strains also include the host of origin in the nomenclature, e.g. A/mallard/Denmark/64650/03(H5N7).
The influenza A virus genome consist of eight negative sense single stranded (ss) ribonucleic acid (RNA) segments packed in the viral core comprised of host cell membrane and a matrix 1 (M1) protein layer. The eight segments are associated with nucleoprotein (NP) and three large proteins; polymerase basic 1 (PB1) and 2 (PB2) protein, and polymerase acidic (PA) protein, which are responsible for RNA replication and transcription. NP encapsulates the RNA and forms ribonucleoprotein (RNP) complexes that protect and stabilise the RNA. Each segment include a sequence of 11-13 nucleotides at the 5′ ends and 9-12 nucleotides at the 3′ ends which are highly conserved and similar for A, B and C viruses. The major glycoproteins HA and NA, and the ionchannel M2 protein, are embedded in a host derived lipid bilayer. Influenza viruses are somewhat pleomorphic in shape, but mostly spherical (80-120 nm in diameter).
All subtypes of influenza A are perpetuated in the wild aquatic bird population, believed to be the natural reservoir of influenza. Under normal circumstances an influenza infection in wild ducks is asymptomatic. The virus replicates in the intestinal tract and is excreted in high concentrations with the faeces for a period up to 30 days. An avian influenza virus can persist in water and retain infectivity for about 100 days at 17° C. and can be stored indefinitely at −50° C. The continuous circulation of influenza A viruses might be due to bird overwintering sites in the subtropics. The 2004 H5N1 strains have become very stable and can survive for 6 days at 37° C. The virus is killed by heat at 56° C. for 3 hours or 60° C. for 30 minutes. Also disinfectants like formalin and iodine compounds efficiently kill the influenza virus. Avian influenza viruses have been believed to be in evolutionary stasis in its natural host, the virus and the host tolerate each other. Generally no severe clinical symptoms are seen when poultry are infected with avian influenza, and the virus is described as a low pathogenic avian influenza virus (LP AIV). The subtypes H5 and H7 have the potential to become highly pathogenic (HP) to chickens through accumulation of mutations after transmission to poultry. Contrary to previous belief, wild migratory birds might play some role in the transmission of HP AIV. Thousands of wild aquatic birds in Hong Kong 2002 and China 2005 became infected with HP AIV H5N1 and this contributed to the spread of HP H5N1 to Europe and Africa in 2005.
Seasonal influenza strains have been isolated from humans and swine all year round, but in temperate climates it is a winter disease probably because people come together and stay in less ventilated rooms due to the cold weather.
Of the 16 recognised subtypes of HA and 9 NAs only H1, H2, H3, N1 and N2 have circulated in humans and swine in the last century. The pandemic introduction in humans of these types were 1918 H1N1, 1957 H2N2 (“Asiatic flu”), 1968 H3N2 (“HongKong Flu”) and non-pandemic introduction of the reassorted new type H1N2 in 2001, respectively. The antigenicity of human influenza viruses are constantly changing by accumulation of mutations in the HA and NA antigenic sites, thereby making the virus capable of evading the host immune system causing epidemics. Viral mutagenesis is enhanced by the lack of “proof reading” in the replication of RNA. The mutation frequency is approximately one in 100,000 nucleotides. At the northern hemisphere seasonal influenza outbreaks usually occur between October and April and from April to October in the southern hemisphere. The antigenic drift of human influenza viruses are closely monitored by the World Health Organization's global influenza surveillance program. The components of the next seasons influenza vaccine for the northern hemisphere is determined in February based on the knowledge about the current circulating strains, and re-evaluated in September for the southern hemisphere.
Antigenic shift can occur in three ways. Either by direct transmission of an avian strain adapted to humans, genetic reassortment or reintroduction of an “old” strain. The possibility of an avian influenza virus crossing the species barrier and infecting humans directly was not recognised before 1997 when 18 people in Hong Kong became ill with HP AIV H5N1.
The origin of the 1918 pandemic is controversial. Taubenberger et at., (Characterization of the 1918 influenza virus polymerase genes. Nature, 2005, 437:889-893) suggested based on phylogenetics of the polymerase genes that the virus was entirely of avian origin. However, there are large disagreements about the actual origin of the virus and many still believe that also this pandemic strain is a reassortant between a mammalian and avian virus most likely occurring from swine. If the virus was of avian origin it might imply that the HP avian viruses circulating currently could cause a new pandemic by direct transmission to humans. Antigenic reassortment occurs when viral segments from two antigenic different viruses infecting the same cell. The reassorted virus contains segments of both strains and if the newly introduced segment is HA (and NA) the complete antigenicity of the virus might change and the virus escapes the host immunity. These reassortants might be catastrophic if the virus is capable of efficient replication in the new host. In worst case such a reassorted strain might lead to pandemics, world-spanning infections to which we have no pre-existing immunity. The pandemics of 1957 and 1968 were reassortants that acquired the HA, NA and PB1 and HA and PB1 genes from an aquatic source, respectively. In 1977 a strain identical to the H1N1 strains that circulated before 1957 re-emerged. Pigs are possible “mixing vessels” for reassorted viruses due to their receptor tropism for both α-(2,3) and α-(2,6) linkage to galactose. Other species like chicken and man might also serve as mixing vessels in the light of direct crossover to humans from an avian source after the discovery of α-(2,3) avian like receptor on cells also in humans and chickens.
The interpandemic evolution of influenza viruses has been thought to be caused by progressive antigenic drift due to the mutability of the RNA genome. H3N2 has been the predominant subtype circulating in humans since 1968 and has been in rapid drift as a single lineage while there has been slow replacement of antigenic variants of the H1N1 viruses. It has been shown that the rate of accumulating mutations is approximately 4-5×10−3 substitutions per nucleotide per year for HA1 others predict a rate of 5.7×10−3 substitutions per nucleotide per year. The HA and NA might evolve independently from each other and reassortments of the internal genes are also known. Positive selection has been inferred on codons involved at antibody antigenic sites, T-cell epitopes and sites important virus egg growth properties. Recent research on viruses has suggested that the evolution of influenza do not always follow a constant rate, but is characterised by stochastic processes, short intervals of rapid evolution, long intervals of neutral sequence evolution and slow extinction of coexisting virus lineages. The evolution seems also more influenced by reassortment events between co-circulating lineage and viral migration than previously believed.
Vaccination is the preferred choice for influenza prophylaxis. Inactivated influenza vaccines are licensed worldwide while cold-adapted live vaccines are licensed only in Russia and the USA. The preferred prophylaxis of annual influenza infections is vaccination with inactivated protein vaccines from virus propagated in hens' eggs. Thus, the common vaccines are the inactivated vaccine viruses which are propagated in hens' eggs and inactivated by formaldehyde or β-propiolactone. There are three classes of inactivated vaccines; whole, split (chemically disrupted with ether or tributyl phosphate) and subunit (purified surface glycoproteins) administrated intramuscularly or subcutaneously. Whole inactivated influenza vaccine is not currently used due to high levels of side effects. The seasonal influenza vaccine (split and subunit) is trivalent, comprising H3N2 and H1N1 influenza A virus strains and an influenza B virus. The normal human vaccine dose is standardised to 15 μg HA protein of each virus component administrated once in normal healthy adults and twice in children and other persons with no pre-existing influenza A immunity. The conventional vaccines induce merely a humoral immune response. The protective effect of the traditional protein split vaccine is very limited and because of the continuous evolution of influenza A virus strains and the typespecific antibodies induced by the conventional vaccines a new vaccine has to be produced every year based on the most recent circulating influenza A strain. Several vaccine improvements are necessary in case of a new emerging human strain. Egg production is too slow (6-12 months) in the case of emerging strains. If this strain is also an AIV virus highly pathogenic (HP) for poultry, egg production might be impossible because the virus kills the egg embryo. Also the availability of eggs might be limited slowdown the vaccine production. In the case of no pre-existing immunity in the population two vaccinations would be necessary, thereby further delaying the vaccine production. Even if there are no new pandemic influenza A among humans but only spread of a HPV AIV among poultry the shortage of eggs will limit production on eggs of traditional seasonal influenza vaccines. In addition, traditional influenza protein vaccines do not have optimal protection as prophylaxis and no therapeutic effect. Thus, there is a need for new alternative influenza vaccines.
Although DNA vaccines were developed more than 16 yeas ago, clinical trials preceding stage I and II in humans are rare. Two veterinary DNA vaccines however, have been licensed; one for West Nile Virus (in horse) and a second for Infectious Hematopoetic Necrosis virus in Salmon. This demonstrates that DNA vaccines can have good protective effects and that new DNA vaccines are not limited by the size of the animal or species. The great success with DNA vaccines observed for the murine model for first generation DNA vaccines did not translate well to humans, nonetheless; researchers have recently demonstrated protective antibodies levels by a single dose of gene gun administrated HA DNA vaccine to humans.
“Nucleic acid immunization” or the commonly preferred name “DNA vaccines” are the inoculation of antigen encoding DNA or RNA as expression cassettes or expression vectors or incorporated into viral vectors with the purpose of inducing immunity to the gene product. Thus, in our definition of DNA vaccines we include all kinds of delivery systems for the antigen encoding DNA or RNA. The vaccine gene can be in form of circular plasmid or a linear expression cassette with just the key features necessary for expression (promotor, the vaccine gene and polyadenylation signal). Delivery systems may most often be naked DNA in buffer with or without adjuvant, DNA coupled to nanoparticles and/or formulated into adjuvant containing compounds or inserted into live viral or bacterial vectors such as Adenovirus, adenoassociated virus, alphavirus, poxviruses, herpes virus etc. DNA vaccines hold great promise since they evoke both humoral and cell-mediated immunity, without the same dangers associated with live virus vaccines. In contrast to live attenuated virus vaccines DNA vaccines may be delivered to same or different tissue or cells than the live virus that has to bind to specific receptors. The production of antigens in their native forms improves the presentation of the antigens to the host immune system. Unlike live attenuated vaccines, DNA vaccines are not infectious and can not revert to virulence. DNA vaccines expressing HA, NA, M, NP proteins or combinations of these have proven to induce immune responses comparable to that of a natural viral infection.
DNA vaccines offer many advantages over conventional vaccines. It can be produced in high amounts in short time, abolishing the need for propagation in eggs, it is cost-effective, reproducible and the final product does not require cold storage conditions, because DNA is stable and resistant to the extremes of temperature. All currently licensed inactivated vaccines are efficient at inducing humoral antibody responses but only live attenuated virus vaccines efficiently induce a cytotoxic cellular response as well.
DNA vaccines induce an immune response which is comparable to the response acquired by natural virus infection by activating both humoral and cell-mediated immunity (6,30). The broad response to DNA vaccines is a result of the encoded genes being expressed by the transfected host cell, inducing both a Th1 and Th2 immune responses. The production of antigens in their native form improves the presentation of the antigens to the host immune system. In contrast, the conventional inactivated influenza protein based vaccines only induce a humoral response (Th2), directed against the influenza surface glycoproteins. This type of response is ineffective against drifted virus variants and therefore the virus composition of the seasonal influenza vaccine has to be assessed every season. Antigenic cross-reactive responses are mainly induced by the more conserved influenza proteins like the nucleoprotein (NP) and the matrix (M) protein. By including these genes in a DNA vaccine higher cross reactivity between drifted and heterologous strains have been shown (4, 7, 8, 13).
Influenza infection and symptoms in ferrets are highly comparable to what is observed in humans and is therefore one of the best models for influenza vaccination trials (22). Influenza HA DNA vaccines in ferrets have also previously proved effective (18,32).
It has previously been shown that 1918 H1N1 whole inactivated virus vaccine induced partly protection against infection with 1918 H1N1 in mice (28), also recently DNA vaccines encoding the HA from 1918 showed complete protection of mice against a 1918 H1N1 challenge (16) but no protection against present day influenza was demonstrated.
We have demonstrated that gene gun administrated codon optimised plasmid DNA vaccine encoding HA and NA with or without M and NP based on the H1N1 pandemic virus from 1918 induce protection in ferrets against infection with a present day H1N1 virus (Bragstad et al 2009 and PCT/DK2008/000201). This demonstrates a vaccine induced protection not mediated by the usual anti-HA and anti-NA antibodies but by a different immunological mechanism most likely involving cellular immunity. Since the internal proteins M and NP encoded by the DNA and/or the influenza A virus are more conserved among different H1N1 strains than HA and NA it can be expected that the induced immunity to NP and M DNA vaccines are more broadly protective which could extend to also new H1N1 strains. The viruses are separated by a time interval of 89 years and differ by 21.2% in the HA1 protein. These results suggest not only a unique ability of the DNA vaccines but also a unique and unexpected feature of the 1918 HA and/or NA in inducing especially broad and efficient protective immunity against even extremely drifted strain variants. The present invention discloses that an induced immune response with a DNA vaccine encoding HA and/or NA of the 1918 H1N1 influenza A gives a high level of cross protection against present day influenza infection.
DNA vaccines do have the ability of immune stimulatory mechanisms. This might be one reason why we observe such a good induced cross reactivity and protection against challenge infection. Cross-protection and cross-reactivity induced by DNA vaccines of strains differing by 11-13% in HA1 has been demonstrated by others (13-15) but not as high as with the 21.2% divergence we observe.
Influenza vaccines that have the ability to induce immune responses able to cross-react with drifted virus variants and even heterologous strains would be of great advantage for both annual vaccine development and in case of emerging new strains.
Since the novel influenza A (H1N1v) is notably different from other human H1N1 viruses it is assumed that present H1N1 virus immunity and seasonal H1N1 vaccines will not induce efficient protection against the novel strain. The novel H1N1v virus is approximately 5% different in the nucleotide sequence from other known swine H1N2 viruses, while it is nearly 24% different from last seasons circulating human H1N1 viruses.
Thus, in the current situation with a new pandemic H1N1v virus and future variants here off we believe the best vaccine for the current circulating viruses and near future viruses will be a DNA vaccine comprising the HA and NA genes of the new pandemic H1N1v virus, the internal genes of the previous pandemic H1N1 virus from 1918 and the HA and NA genes of the circulating seasonal H3N2 virus. The genes of the H1N1v strain will be included to induce perfect protection against the circulating H1N1v and for future variants of this strain. We expect this strain with variants hereof to be the next seasonal H1N1 viruses in humans. The NP and M internal genes of the 1918 H1N1 pandemic is included as these are the ancestors of all other NP and M genes in human influenza viruses. These highly conserved genes are expected to induce better cross-protection. The HA and NA genes of the seasonal H3N2 virus are included as these will give the best protection against currently circulating H3N2 viruses and future H3N2 viruses. We believe that these gene combinations in a DNA vaccine will be the optimal more universal influenza vaccine at present time and for the nearest future.
We have surprisingly shown that a mix of DNA plasmids of both “initial” pandemic genes with few or no glycosylation sites and optionally present time genes induces the ultimate protection in the sense that the more antigenic sites will be exposed to the host immune system for antibody recognition. Alternatively influenza genes where all or some of the glycosylation codons have been changed/removed so the protein expressed has less or no glycosylation sites can be used in a mixture either themselves or with pandemic influenza DNA to make up a universal influenza vaccine.